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J. Dairy Sci. 89:3626-3635
© American Dairy Science Association, 2006.

Association of Toll-Like Receptor 4 Polymorphisms with Somatic Cell Score and Lactation Persistency in Holstein Bulls

B. S. Sharma, I. Leyva, F. Schenkel and N. A. Karrow1

Centre for Genetic Improvement of Livestock, Department of Animal and Poultry Science, University of Guelph, Guelph, N1G 2W1, Canada

1 Corresponding author: nkarrow{at}uoguelph.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Mastitis, an inflammatory disease of the mammary gland generally caused by intramammary infections, is the most frequently occurring disease in the North American dairy industry. Reduced milk yield, milk quality, and lactation persistency as well as early culling contribute to the economic losses associated with this disease. During intramammary infections, cells of the innate immune system become activated through pattern recognition receptors that recognize conserved molecular signatures associated with the invading pathogen. The quality, timing, and intensity of the host inflammatory and subsequent immune response determine the fate of this disease. Toll-like receptor 4 (TLR4) is an important pattern recognition receptor that recognizes endotoxins associated with gram-negative bacterial infections. Its role in pathogen recognition and subsequent initiation of the inflammatory and immune response makes it a suitable candidate gene for enhancing disease resistance in Canadian Holsteins. In this study, polymorphisms in the TLR4 gene were identified in the Canadian Holstein bull population. Genotypes and haplotypes were constructed, and their associations with somatic cell score and lactation persistency were determined. Sequencing of selective DNA pools was used to reveal polymorphisms in TLR4. Two DNA pools were constituted based on high and low estimated breeding values for somatic cell scores. A total of 3 single nucleotide polymorphisms (SNP), including 1 SNP in a putative promoter region (P-226) and 2 SNP in exon3 (E3+1656 and E3+2021) of TLR4 were detected. A total of 388 bulls were genotyped for the SNP, haplotypes were reconstructed, and their frequencies were obtained. Polymorphisms in these regions were found to be associated with estimated breeding values for lactation persistency, and somatic cell scores in the Canadian Holstein bull population. The unfavorable alleles at P-226 and E3+1656 were found at a frequency of 40 and 37%, respectively; hence, selection against these alleles is promising in Canadian Holsteins. Selection against the unfavorable allele, T at E3+2021, is limited because of its low frequency (7%). Two frequently occurring haplotypes (GCC and CTC) occurred in 86% of the Canadian Holstein bull population chosen for genotyping. The most frequent haplotype (GCC; 54%) was found to be associated with higher lactation persistency and lower somatic cell scores. The transversion SNP in the putative promoter region (P-226) was in a potential DNA binding site.

Key Words: Toll-like receptor 4 • health trait • somatic cell score • Canadian Holstein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Mastitis is an inflammatory disease of the mammary gland that is generally caused by IMI. It is the most frequently occurring disease in North American dairy cows, and economic losses associated with this disease are attributed to reduced milk yield and quality, reduced lactation persistency, and early culling (Fetrow, 2000; Capuco et al., 2003). During IMI, mammary epithelial cells and tissue macrophages become activated via their pattern recognition receptors, which recognize molecular signatures associated with the invading pathogen, called pathogen-associated molecular patterns (PAMP). Pattern recognition receptor–PAMP ligation induces the transcription and secretion of cytokines and chemokines that elicit first the recruitment of blood neutrophils and then monocytes to the site of infection. These cells are the effector cells of the innate immune system and are involved in the inflammatory response and in regulating the subsequent immune response.

The Toll-like receptors (TLR) are a multigene family of pattern recognition receptors that are members of the TLR–interleukin 1 superfamily. These receptors recognize a great variety of PAMP, and therefore play a central role in the initiation of inflammatory response and subsequent adaptive immune response to microbial pathogens (Sabroe et al., 2003; Takeda et al., 2003). Currently, at least 13 members of the TLR family have been identified in mammals; genes encoding 10 of these receptors have recently been mapped to the bovine genome (McGuire et al., 2006).

Each member of the TLR family is capable of recognizing a unique set of PAMP. Lipopolysaccharide endotoxin from Escherichia coli and other gram-negative bacteria, for example, are recognized by TLR4. Toll-like receptor 4 also recognizes PAMP associated with Mycobacterium tuberculosis, Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans (Poltorak et al., 1998; Lien and Ingalls, 2002; Netea et al., 2002), and host inflammagens such as heat-shock protein (Hsp60), fibrinogen, fibronectin, and oligosaccharides of hyaluronan (Lien and Ingalls, 2002). Peptidoglycan and lipoteichoic acid from Staphylococcus aureus and other gram-positive bacteria are uniquely recognized by TLR2 (Bannerman et al., 2004). Recent evidence showing that concentrations of important facilitators of TLR2 and TLR4 signaling, specifically soluble CD14 and LPS-binding protein, are increased in milk during E. coli and Staph. aureus IMI (Bannerman et al., 2004). That the TLR2 and TLR4 genes (but not the TLR9 gene) are strongly expressed during mastitis caused by Staph. aureus (Goldammer et al., 2004) suggests that TLR2 and TLR4 may play a role in the host response to IMI.

Polymorphisms in genes encoding receptors associated with the innate immune system are likely to contribute to the overall variation in the resistance or susceptibility to mastitis in dairy cattle. Polymorphisms in the CXCR2 chemokine receptor gene, for example, have recently been associated with mastitis susceptibility and neutrophil function (Youngerman et al., 2004; Rambeaud and Pighetti, 2005). Additionally, polymorphisms in BoLA-DR3 have been associated with clinical mastitis and may play critical role in altered antigen-binding affinity to the major histocompatibility complex class II molecule and recognition of the peptide by T cells (Sharif et al., 2000).

Several lines of evidence suggest that lack of, or polymorphism in, TLR4 can compromise immune responses to certain pathogens. When TLR4–/ – knockout mice were challenged with LPS, for example, they were unable to mount a cellular response, and no detectable change in tumor necrosis factor-{alpha} was observed (Hoshino et al., 1999). Polymorphisms in the coding and promoter regions of TLR4 can also determine different host resistance or susceptibility patterns to various infectious diseases. A recently described association between the Asp299Gly polymorphism in human TLR4 and gram-negative–induced septic shock suggests that a functional defect in TLR4 leads to an increased susceptibility to gram-negative bacteremia (Lorenz et al., 2002). The same mutation is also associated with hypo-responsiveness to LPS in humans (Arbour et al., 2000). Last, analysis of the truncated and mutated promoter sequence of TLR4 in mice has revealed several positive and negative regulatory elements considerably affecting promoter activity and TLR4 mRNA levels (Roger et al., 2005).

Given that TLR4 is involved in PAMP recognition, mutations in TLR4 can compromise the host immune response to certain pathogens, that because TLR4 is highly polymorphic in the bovine species (White et al., 2003), and TLR4 expression is associated with IMI, this gene may be a potential candidate for use in marker-assisted selection to enhance mastitis resistance in dairy cattle. Therefore, the intent of this study was to reveal the 5'-upstream promoter sequence to detect single nucleotide polymorphisms (SNP) in the putative promoter region of TLR4, and to evaluate the possible association of SNP in the coding and promoter regions with health-related traits such as SCS and persistence of lactation in Canadian Holsteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Resource Population
A total of 388 bull semen samples were selected from a resource population of 2,166 Holstein bulls. The EBV of these bulls were obtained from a national genetic evaluation database generated in October 2003 by the Canadian Dairy Network. Among the 388 bulls, a total of 48 bulls were selected that were among the most frequently used sires in an Ontario sentinel herd study (Kelton et al., 1999). The remaining 340 bulls were selected within families on the basis of extreme EBV for either protein yield or SCS. These bulls were the progeny of 30 sires. The majority of the selected population was composed of half-sib families of 20 sires, the size of which ranged from 2 to 30 bulls. Semen samples were kindly provided by Semex Alliance (Guelph, Ontario, Canada).

In the present study, the health-related traits considered in the analyses were SCS and LP because QTL for SCS exist on Bos taurus autosome 8 (BTA8). Somatic cell score represents a log score of the milk SCC and has been genetically correlated with clinical mastitis (Shook and Schutz, 1993). Macrophages are the dominant somatic cell population found in uninfected milk and represent a primary line of defense against IMI (Lee et al., 1980; Sordillo and Nickerson, 1988). Lactation persistency is defined as the expected milk yield on d 280, expressed as a percentage of the average actual base population yield on d 60 (Jamrozik et al., 1998). Animals with a greater LP are generally less susceptible to metabolic disorders, health problems, and fertility problems (Dekkers et al., 1998), and clinical mastitis can increase mammary epithelial cell apoptosis, thereby reducing LP (Capuco et al., 2003). The EBV for SCS and LP were available for 387 and 359 bulls, respectively.

DNA Isolation
The DNA was extracted from semen samples following the standard phenol-chloroform method with minor modifications (Winfrey et al., 1997). The DNA concentration and quality were assessed based on the absorbance of UV light at 260 (A260) and 280 nm (A280) with an Eppendorf BioPhotometer (Berlin, Germany).

Determination and Analysis of the 5'-Upstream Sequence to Exon 1 of TLR4
The 5'- flanking sequence upstream of exon1 of TLR4 was obtained using PCR and adaptor-ligated genomic B. taurus DNA restriction enzyme fragment libraries, generated using the Universal GenomeWalker Kit (BD Biosciences Clontech, Palo Alto, CA). Briefly, 4 separate walker libraries were constructed by ligating a specially designed adapter sequence to bovine genomic DNA (BD Biosciences Clontech), and each was digested by 4 different restriction enzymes (DraI, EcoR V, PvuII, and StuI). The gene-specific nested primers were designed from the TLR4 exon1 and available upstream sequence (GenBank accession no. AY297041). The major distinct PCR products were then sequenced by gene-specific primers (GSP). An initial ~800-bp fragment was amplified by 2 rounds of nested PCR using the library made with StuI. The 2 GSP used were TLR4W0 (5'-AGC ATG ACC CTC TGT CTG TGC CGG CCA G-3') and TRL4W1 (5'-CCG GCC AGG GCA GCC TCC GAG GC-3'). The first round of PCR was 2 min at 94°C, followed by 30 s at 94°C and 3 min at 68°C for 35 cycles, and ending at 68°C for 7 min. The second round of PCR was 10 min at 94°C, followed by a touchdown profile. The initial 7 cycles were 30 s at 94°C, 30 s at 68°C, and 2 min at 72°C, followed by 30 cycles for 30 s at 94°C, 30 s at 61°C, and 2 min at 72°C for 30 cycles, and a final extension of 10 min at 72°C. Gene-specific nested primers were designed from the newly obtained sequence and were used for subsequent walking. Similarly, an additional 1.3-kb upstream sequence was amplified by 2 additional rounds of walking. Finally, using genomic bovine DNA as a template, a total of 2,112 bp of TLR4 promoter region was amplified and sequenced.

Construction of DNA Pools for SNP Detection
Twenty bulls with high EBV for SCS and 20 bulls with low EBV for SCS were selected for creating DNA pools. DNA quantification of these samples was further reassessed using the PicoGreen dsDNA quantification procedure (Molecular Probes; Invitrogen, Carlsbad, CA) on a Victor 3 fluorescent plate reader (PerkinElmer, Wellesley, MA). Two DNA pools were constructed by aliquoting an equal amount of DNA from each selected bull in each group. These pools were used for the amplification of 3 exons of TLR4 and the newly obtained 5'-upstream putative promoter region of TLR4 to detect SNP. Three SNP were detected on the sequencing electropherograms (Figure 1Go), including one SNP at the 226-bp position in the putative promoter region (P-226) and 2 SNP in exon3 of TLR4 at the 1,656-bp (E3+1656) and 2,021-bp (E3+2021) positions.


Figure 1
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  		Figure 1. Diagram showing 3 single nucleotide polymorphisms (SNP) on sequencing electropherograms of DNA pools in the TLR4 gene.

 
SNP Genotyping
The tetra-primer Amplification Refractory Mutation System–PCR procedure (Ye et al., 2001) was used to genotype the 3 SNP. This is a simple and economical method involving a single PCR reaction. The method uses 4 primers (Table 1Go) to amplify a larger control fragment from a DNA sequence containing a given SNP, producing amplicons representing each of the 2 allelic forms (Figure 2Go). In this method, a mismatch is deliberately introduced at the 3' end of each of the 2 allele-specific primers to increase the specificity of the reaction. The primers can be designed to amplify fragments of differing sizes corresponding to each allele to enable easy resolution of the allele-specific bands on an agarose gel. For SNP genotyping, primers were designed using the primer design computer program made accessible by Ye et al. (2001) (http://cedar.genetics.soton.ac.uk/public_html/primer1.html). The method proved to be reliable, inexpensive, and easy to use. The PCR was performed in a total volume of 10 µL, containing 30 ng of template DNA, 10 pmol of each inner primer, 1 pmol of each outer primer, 200 µM deoxyribonucleotide tri-phosphates, 2.5 mM MgCl2, 1 x PCR buffer, and 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). The touchdown profile was followed using 6°C higher than the annealing temperature in the first PCR cycle, subsequently decreasing the annealing temperature by 1°C per cycle in the following 6 cycles, and then keeping the annealing temperature at the final level thus obtained for the remaining 30 cycles. The PCR profile was as follows: 94°C for 10 min, 36 cycles of 30 s at 94°C, 30 s of annealing temperature (with touchdown profile), and 1 min of extension at 72°C, ending with 5 min at 72°C. The annealing temperatures were 62, 65, and 62°C for P-226, E3+1656, and E3+2021 SNP, respectively. The tetra-primer Amplification Refractory Mutation System–PCR products were observed on a 1.5% agarose gel stained with ethidium bromide.


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  		Table 1. The tetra-primer Amplification Refractory Mutation System–PCR primers and conditions for genotyping TLR4 single nucleotide polymorphisms
 

Figure 2
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  		Figure 2. An illustration of single nucleotide polymorphism (SNP) typing using the technique of tetra-primer Amplification Refractory Mutation System–PCR. Diagram shows genotyping of E3+1656 SNP. Four primers were used: The 2 outer primers amplify a control fragment (416 bp) of the gene that contains E3+1656 SNP. The inner primers were designed to amplify the 2 different allelic fragments of 269 and 202 bp representing the C and T alleles, respectively.

 
Haplotype Construction
The haplotype probabilities were reconstructed using algorithm and HAPROB software (Boettcher et al., 2004). This software provides probabilities of haplotype combinations for members of half-sib families. The procedure involves 2 steps and requires genotypes only from siblings, not from parents. In the first step, haplotype probabilities of the sires were determined based on the genotypes of the offspring and their allelic frequencies in the general population. In the second step, the haplotype probabilities of the offspring were calculated based on the sire haplotype frequencies. These 2 steps were alternated iteratively until the estimated population frequencies converged to stable values. The final results were a set of estimated haplotype probabilities for each animal, expressed as the expected number of copies of each haplotype carried by each animal.

Genotype Analyses
Associations of 3 SNP of the TLR4 gene with traits of interest were estimated individually, considering the genotype of SNP as a fixed effect. The following mathematical model was used and analyzed by SAS software (SAS Institute, 1999):


Formula

where EBVij is trait EBV of the jth animal with the ith genotype, µ is the overall mean, Gi is the fixed effect of the ith genotype of TLR4 SNP, and eij are random residual effects.

Haplotype Analyses
Haplotype effects were estimated by regressing EBV on haplotype probabilities, which are expressed as the expected number of copies of each haplotype. Three haplotypes were rare (joint frequency = 0.02) and were therefore pooled together (i.e., HapP) into a single common effect. The following model was used for statistical analyses using SAS software (SAS Institute, 1999):


Formula

where EBVij is trait EBV of the jth animal, µ is the overall mean, ßi is the linear regression coefficient for the ith haplotype, Hapij is the probability of the ith haplotype of the jth individual, h is the number of haplotypes evaluated, and eij are random residual effects.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
TLR4 Promoter Sequence
The 5'-upstream promoter region of the TLR4 gene was sequenced using the genome walker technique (Clontech). After a total of 3 walking steps, a 2.1-kbp sequence in the promoter region of TLR4 was obtained upstream of the translation start site. This sequence was compared with recently available sequence information from GenBank and was found to be 100% identical to the B. taurus chromosome Un genomic contig sequence (GenBank accession no. NW_482804) obtained by whole genome shotgun sequencing and released in September 2004 by the Baylor College of Medicine Human Genome Sequencing Center.

Potential DNA binding sites within the 700-bp region of the TLR4 promoter sequence were identified by web-based computer analysis (htpp://www.motif.genome.ad.jp). Several DNA binding sites were revealed by this analysis, including sites for c-Ets-1 (p54), AP-1, AP-4, MZF1, ADR1, GATA-1, GATA-2, GATA-3, Oct-1, and CRE-binding protein. A TATA box, CCAAT sequences, and GC-rich regions were lacking in this region (Figure 3Go).


Figure 3
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  		Figure 3. Bovine TLR4 5'-flanking region sequence (–700 bp), and putative binding sites for bovine transcription factors (overlined). The start codon is localized at +1. Transversion single nucleotide polymorphism (SNP; G/C) P-226 was represented by bold letters. (Oct = octamer factor; NF-AT = nuclear factor of activated T cells; GATA = GATA-binding factor; CREB = cAMP-responsive element-binding protein; STRE = stress-response element; AP = activator protein; ADR1 = alcohol dehydrogenase gene regulator 1; GCR = glucocorticoid receptor; MZF1 = myeloid zinc finger gene 1; CF = chorion factor).

 
SNP Detection
A total of 3 SNP were detected, including a transversion C {iff} G SNP at position –226 bp in the putative promoter region (P-226) and 2 transition C {iff} T SNP at position 1,656 bp in exon3 (E3+1656) and position 2,021 bp in exon3 (E3+2021; Figure 1Go). The novel P-226 SNP was submitted to the National Center for Biotechnology Information (accession no. ss49839679), which is released in the dbSNP Build 126. This SNP is in a binding site of c-Ets-1, MZF1, and ADR1 transcription factors (Figure 3Go).

Genotypic and Allelic Frequencies of TLR4 SNP in Canadian Holstein Bulls
The genotypic frequencies of CC, CG, and GG at P-226 were observed as 58 (15%), 193 (50%), and 137 (35%), respectively. The frequency of allele C was 40%, compared with 60% of allele G. At position E3+1656, the frequency of the CC, CT, and TT genotypes were 144 (37%), 200 (52%), and 44 (11%), respectively. The C allele was predominant over the T allele (63 vs. 37%). The largest differences in genotype frequencies were shown by SNP E3+2021. For this SNP, genotype TT was found at a very low frequency (3, 1%), whereas the majority of the population was either homozygous for CC (338, 87%), or heterozygous (47, 12%). The T allele was rare compared with the C allele (7 vs. 93%).

The frequencies of genotypes were in agreement with the Hardy–Weinberg equilibrium (Falconer and Mackay, 1996) within all 3 SNP (the probabilities of the {chi}2 tests for deviation from the equilibrium were P = 0.75, P = 0.12, and P = 0.64 for P-226, E3+1656, and E3+2021, respectively). When considering 2 SNP together, the equilibrium in genotypic frequencies was tested by a {chi}2 test of expected and observed frequencies of gametic types (Falconer and Mackay, 1996). All pairwise combination tests showed a significant disequilibrium (P < 0.01).

Genotype Analyses
The focus of the genotype analyses was on finding associations of TLR4 polymorphisms with health-related traits, in particular, SCS and LP. Lactation persistency was influenced by genotypes of P-226 (P < 0.004), E3+1656 (P < 0.04), and E3+2021 (P < 0.002). Somatic cell score was associated with the E3+2021 genotype (P < 0.03).

The least squares means for the P-226, E3+1656, and E3+2021 genotypes for LP and SCS are presented in Table 2Go. Genotypes GG and CG of P-226 were highly associated with increased LP. Similar findings were observed at E3+1656, where allele C was associated with higher LP and genotypes CC and CT had higher LP than the genotype TT. Only 3 animals had the genotype TT, for the E3+2021 SNP. Since these were excluded from the analyses, only solutions for the genotypes CC and CT were obtained. The C allele of E3+2021 was associated with higher LP and lower SCS. This nonsynonymous SNP changes an AA from Thr (C allele) to Ile (T allele) in the predicted transmembrane–cytoplasmic domain (White et al., 2003). The estimated differences between homozygous CC and heterozygous genotypes were 1.4% and –0.1 points for LP and SCS, respectively, which corresponds to 0.52 and –0.40 SD units of the respective traits.


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  		Table 2. Least squares means ( ± SE) for the genotypes of single nucleotide polymorphisms (SNP) in the TLR4 gene for lactation persistency (LP) and SCS
 
Haplotype Construction
All 3 SNP were used for haplotype reconstruction. The estimated frequencies of all the haplotypes are presented in Table 3Go. The most common haplotype among Holstein bulls was GCC (Hap3), with an estimated frequency of 0.54. More than 86% of the haplotypes present were either GCC (Hap3) or CTC (Hap4). The rest of the 6 haplotypes had a combined estimated frequency of about 14%. Two haplotypes, Hap5 and Hap6, both bearing a T allele at E3+2021, were rare (<0.005%) because this allele is at a very low frequency (7%) in the population.


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  		Table 3. Probabilities of single nucleotide polymorphism (SNP) haplotypes (Hap) at the TLR4 gene in Canadian Holstein bulls
 
Haplotype Effects
Because haplotypes 5, 6, and 7 together represented only 2% of all haplotypes, they were pooled (HapP) as a single effect for further analyses. Following this pooling, the linear effect of the 6 haplotypes was estimated. These haplotypes had effects on both LP and SCS that were also found to be associated with various SNP. The effect of the most common haplotype (Hap3) was used as a control, and rest of the haplotypes were contrasted against this haplotype.

Hap1, Hap4, and HapP were found to be significantly different from Hap3 for LP, whereas Hap1 was different for SCS also. However, Hap2 and Hap8 were not different from Hap3 for any of these traits, although differences were observed for LP at a lower level of significance (Table 4Go). Interestingly, Hap3 was superior in terms of both traits when compared with other haplotypes. The second most common haplotype, Hap4, which represents approximately 32% of the population, showed 0.51% less LP as compared with Hap3. Haplotype 4 was found to carry the C allele at P-226 and the T allele at E3+1656, whereas at both the positions, Hap3 showed the alternative alleles. In agreement with the genotype analyses, the G allele at P-226 was associated with increased LP as compared with the C allele. A similar finding was also obtained for the C allele at E3+1656 when compared with the T allele.


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  		Table 4. Contrast between haplotypes (Hap) and the most frequent haplotype (Hap3) for lactation persistency (LP) and SCS1
 
Haplotype 1 was significantly different from Hap3 for LP and SCS. For LP, to some extent the observation was in agreement with genotype analyses, since both of these haplotypes carried different alleles for P-226, which are associated with LP differences. In the genotype analyses, E3+2021 was found to be associated with SCS; however, both the haplotypes shared the same allele for this SNP. In the genotype analyses, the T allele for E3+2021 was found to be associated with higher SCS; however, Hap8 composed of the T allele at this location combined with the C allele for P-226 was not different from Hap3 in terms of SCS. The observations obtained from both of these comparisons (Hap1 vs. Hap3 and Hap8 vs. Hap3) suggest that SNP in the putative promoter region might have an effect on SCS. Haplotype P (Hap5, Hap6, and Hap7), which carries a T allele for E3+2021 with either allele for P-226, also showed an increased SCS at slightly lower significance (P < 0.07). The observed differences between genotype analyses and haplotype analyses for SCS cannot be compared as such because the contrast between haplotype effects estimates only the additive linear effect of alleles (Schenkel et al. 2005).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The bovine TLR4 putative promoter region was sequenced for 2.1 kbp located upstream of the first exon of TLR4 and screened for DNA polymorphisms. In addition, all 3 exons of bovine TLR4 were screened for DNA polymorphisms. Three SNP were detected in these regions and were evaluated for their association with health-related traits by genotype and haplotype analyses.

Sequence analysis of the 5'-upstream TLR4 promoter sequence revealed several potential DNA binding sites, including c-Ets-1 (p54), AP-1, AP-4, MZF1, ADR1, GATA-like binding factors, Oct-1, and CRE-binding protein. Conversely, the region lacked a TATA box, a CCAAT box sequence, or a GC-rich region. These results were compared with the promoter region of mice (GenBank accession no. AF177767) and humans (Gen-Bank accession no. AF177765). Sequence analyses of the 5'-upstream 500 bp of mouse TLR4 and 400 bp of human TLR4 promoter regions also showed consensus-binding sites for the Ets family of transcription factors, octamer-binding factors, and the absence of a TATA box or a CCAAT box sequence (Rehli et al., 2000). Similar findings were also observed 600 bp 5'-upstream of the putative promoter region in porcine TLR4 (Thomas et al., 2005).

The SNP detection was carried out by sequencing pooled DNA from bulls with extreme EBV for SCS. We successfully detected 3 SNP, 2 of which were in exon3 at locations 1,656 bp (E3+1656) and 2,021 bp (E3+2021). Both of these SNP were also reported in Bos taurus taurus (White et al., 2003). Exon1 and exon2 were found to be nonpolymorphic based on the sequencing of DNA pools constructed in this study. One SNP was discovered in the putative promoter region at position 266 bp 5'-upstream of the TLR4 gene (P-226). After individual genotyping of each SNP, we observed that the frequencies of the C allele were 45, 57.5, and 90% in the high-SCS pool and 42.5, 55, and 87.5% in the low-SCS pool for P-226, E3+1656, and E3+2021, respectively. It is quite possible that other SNP remain undetected because identification was based on 2 DNA pools composed of 40 animals belonging to either high or low EBV for SCS.

In this study, P-226 SNP was found to be associated with LP, where the C allele was associated with a lower LP as compared with allele G. In fact, this SNP (G -> C) was in a potential DNA binding site either for c-Ets-1 (p54), or MZF1, or the ADR1 transcription factor (Figure 3Go). Haplotypes constituted by the C allele also showed higher SCS, which is an indication of higher susceptibility to clinical mastitis (Philipsson et al., 1995). Hence, we hypothesized that this SNP is functional and may have an impact on the transcriptional level of TLR4 mRNA.

Genotype and haplotype analyses revealed that these SNP have effects on LP and SCS. Persistency of lactation generally refers to the rate of decline in daily milk yield after the peak yield. A cow with a flatter lactation curve is considered to be more persistent than a cow with the same total yield, but with a curve that decreases rapidly after the peak yield. Persistency has direct economic value because persistent cows are more efficient at utilizing roughage, suffer less metabolic stress because of high peak yield, and are thus more disease resistant (Dekkers et al., 1996; Solkner and Fuchs, 1987). It is noticeable that the most frequent haplotype (Hap3) composed of favorable alleles at all locations (GCC), was more persistent compared with the other haplotypes. The next most frequent haplotype, Hap4 (32%), was associated with low LP, which suggests that selection against this haplotype may be promising to improve this trait in Canadian Holsteins. In the studied population few of the rare haplotypes were found, possibly because these haplotypes may not have actually existed in the population, even though they were identified as plausible haplotypes for some individuals by the reconstruction program (Boettcher et al., 2004).

The association of the TLR4 polymorphism with SCS may be attributed to the high genetic correlation between SCS and occurrence of clinical mastitis (Shook and Schutz, 1993.) Somatic cell counts are currently used as an indicator of mastitis and as a tool for selection for mastitis resistance (Koivula et al., 2005). It is also possible that the association between SNP in TLR4 and SCS and LP may be due to linkage with other gene(s) that truly influence these traits, because linkage disequilibrium extends over large distances in the North American Holstein cattle population (Vallejo et al. 2003). TLR4, for example, has been mapped to the distal end of BTA8 (White et al., 2003) and QTL affecting SCS, and clinical mastitis has also been found on this chromosome. Klungland et al. (2001) found a single QTL affecting SCC localized at position 54 cM, and a QTL for clinical mastitis at position 46 cM in one Norwegian sire family. Although QTL for LP have not been identified, clinical mastitis can increase mammary epithelial cell apoptosis, thereby reducing LP (Capuco et al., 2003). Our observation that the unfavorable CT genotype of SNP E3+2021, which was associated with high SCS and low LP, supports this hypothesis. An analysis of an independent cattle population may help to determine whether these associations are truly attributed to linkage disequilibrium (Vallejo et al., 2003).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
In conclusion, a total of 2.1 kbp 5'-upstream putative regulatory sequence of TLR4 was revealed. Three SNP, including one at the putative promoter region (P-226) and 2 in exon3 (E3+1656 and E3+2021) of the TLR4 gene were detected through sequencing of extreme DNA pools established on EBV for SCS. These SNP were found to be associated with LP and SCS. After confirmation of these effects on molecular mechanisms of immune function and disease resistance underlying these traits, marker-assisted selection for P-226 and E3+1656 may be a promising strategy for improving these traits. Haplotype frequencies showed that 2 haplotypes contributed 86% in the population; however, both were significantly different for their effects on LP. These haplotypes should be further assessed for immune function and disease resistance prior to their practical application. The SNP at the 5'-upstream sequence (P-226) is in a potential DNA binding site; hence, we hypothesize that it may alter gene function. Studies in this area are warranted.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
The financial support of the Natural Sciences and Engineering Research Council, DairyGen, and Dairy Farmers of Ontario are gratefully acknowledged.

Received for publication December 14, 2005. Accepted for publication May 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


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